Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have shown that the HIV-1 laboratory strain NL4-3 that contains P236L [a reverse transcriptase mutation conferring resistance to the nonnucleoside reverse transcriptase inhibitor (NRTI) delavirdine] replicates more slowly than wild-type NL4-3. Other NNRTI-resistance mutations, such as K103N and Y181C, do not reduce the replication capacity of NL4-3 as much as P236L and develop more frequently in HIV-1 isolates from patients failing delavirdine. However, a minority of patients on delavirdine therapy still have isolates with P236L. We postulated that reverse transcriptase (RT) sequences from these patient isolates contain other mutations that compensate for the adverse effect of P236L. To test this hypothesis, we created 15 chimeric NL4-3 isolates that contained delavirdine-resistant RT sequences derived from eight patient isolates and characterized their replication kinetics. Nine of 10 patient-derived clones containing P236L replicated as slowly as NL4-3 with P236L. In contrast, three of five clones that did not have P236L (but had either K103N or Y181C) replicated significantly better than NL4-3 with P236L. Thus, the majority of patients who acquire P236L during delavirdine therapy do not have RT mutations that compensate for the replication defect conferred by P236L. We hypothesize that HIV-1 isolates with P236L may have a compensatory mutation outside RT. Alternatively, variants of HIV-1 with reduced replication fitness may be selected during antiretroviral therapy, suggesting that stochastic events rather than viral replication fitness may determine which drug-resistant mutants emerge early during antiretroviral failure. In some isolates, it appears that the background RT sequence can contribute significantly to the replication fitness of drug-resistant HIV-1 variants.
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PMID:Impact of clinical reverse transcriptase sequences on the replication capacity of HIV-1 drug-resistant mutants. 1143 54

In an effort to elucidate a set of structure-activity relationships in the alkenyldiarylmethane (ADAM) series of non-nucleoside reverse transcriptase inhibitors, a number of modifications were made at two locations: (1) the meta positions of the two aromatic rings and (2) the end of the alkenyl chain. Forty-two new ADAMs were synthesized and evaluated for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cell culture and for inhibition of HIV-1 reverse transcriptase. The size of the aromatic substituents was found to affect anti-HIV activity, with optimal activity appearing with Cl, CH(3), and Br substituents and with diminished activity occurring with smaller (H and F) or larger (I and CF(3)) substituents. The substituents at the end of the alkenyl chain were also found to influence the antiviral activity, with maximal activity associated with methyl or ethyl ester groups and with diminished activity resulting from substitution with higher esters, amides, sulfides, sulfoxides, sulfones, thioesters, acetals, ketones, carbamates, ureas, and thioureas. Twelve of the new ADAMs displayed submicromolar EC(50) values for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cells. Selected ADAMs, 19 and 21, were compared to previously published ADAMs 15 and 17 for antiviral efficacy and activity against the HIV-1 reverse transcriptase enzyme. All four ADAMs were found to inhibit HIV-1 reverse transcriptase enzyme activity, to inhibit the replication of a variety of HIV-1 clinical isolates representing syncytium-inducing, nonsyncytium-inducing, and subtype representative isolates, and to inhibit HIV-1 replication in monocytes. Subsequent assessment against a panel of site-directed reverse transcriptase mutants in NL4-3 demonstrated no effect of the K103N mutation on antiviral efficacy and a slight enhancement (6- to 11-fold) in sensitivity to AZT-resistant viruses. Additionally, ADAMs 19 (44-fold) and 21 (29-fold) were more effective against the A98G mutation (found in association with nevirapine resistance in vitro), and ADAM 21 was 5-fold and 2-fold more potent against the Y181C inactivation mutation than the previously reported ADAMs 15 and 17, respectively. All four ADAMs were tested for efficacy against a multidrug-resistant virus derived from a highly experienced patient expressing resistance to the reverse transcriptase enzyme inhibitors AZT, ddI, 3TC, d4T, foscarnet, and nevirapine, as well as the protease inhibitors indinavir, saquinavir, and nelfinavir. ADAM 21 was 2-fold more potent than ADAM 15 and 6-fold more potent than ADAMs 17 and 19 at preventing virus replication. Thus, we have identified a novel series of reverse transcriptase inhibitors with a favorable profile of antiviral activity against the primary mutation involved in clinical failure of non-nucleoside reverse transcriptase inhibitors, K103N, and that retain activity against a multidrug-resistant virus.
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PMID:The biological effects of structural variation at the meta position of the aromatic rings and at the end of the alkenyl chain in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors. 1170 13

Viral infection triggers a cascade of interferon response genes, but the mechanisms that prime such innate antiviral defenses are poorly understood. Among candidate cellular mediators of the antiviral response are the double-stranded RNA (dsRNA)-binding proteins. Here we show that a C-terminal variant of the ubiquitous dsRNA-binding protein, nuclear factor 90 (NF90ctv), can activate the interferon response genes in the absence of viral infection. NF90ctv-expressing cells were infected with the syncytium-inducing HIV-1 strain NL4-3 and were shown to inhibit viral replication. To gain insight into this mechanism of protection, we analyzed the expression profiles of NF90ctv-positive cells as compared with parental cells transduced with the empty vector. Of the 5600 genes represented on the expression arrays, 90 displayed significant (4-fold or more) changes in mRNA levels in NF90-expressing cells. About 50% are known interferon alpha/beta-stimulated genes. The microarray expression data were confirmed by quantitative reverse transcriptase-polymerase chain reaction analysis of six representative interferon-inducible genes. Electrophoretic mobility shift assays showed that the biological response is mediated by the activation of transcription factors in NF90ctv-expressing cells. Functional significance of the activated transcription complex was evaluated by transfection assays with luciferase reporter constructs driven by the interferon-inducible promoter from the 2'-5'-oligoadenylate synthetase (p69) gene. Resistance to HIV-1, caused by the expression of NF90ctv in the cell culture system, appears to be mediated in part by the induction of interferon response genes. This leads to a hypothesis as to the mechanism of action of NF90 in mediating endogenous antiviral responses.
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PMID:Nuclear factor 90 mediates activation of the cellular antiviral expression cascade. 1203 89

Infection of CD4- cells by HIV-1 is well documented, but the mechanism responsible remains a matter of discussion. Previously we modified an HIV-1 virus strain, NL4-3, by deleting the Env proteins (gp41 and gp120) and inserting the enhanced green fluorescent protein (EGFP), and found that the Env(-) virus infects several types of CD4- cells. Here, we have prepared Env(-) virus from both the CD4- cell line, 293T, and the CD4+ cell lines, CEM and SUPT1, and found that HIV-1 Env(-) virus from either cell type is infectious for both CD4+ and several CD4- cell lines. Replication of HIV-1 Env(-) virus-infected cells was demonstrated by p24 gag protein assays and real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the culture medium from infected cells. Virus collected from the HIV-1-Env(-) infected cultures proved infectious to several CD4- cell lines. Our results suggest that HIV-1 infects both CD4- and CD4+ cells using a gp120-independent mechanism. This infection mechanism may provide new explanations for HIV-1 latency and persistent infection in patients.
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PMID:gp120-Independent infection of CD4(-) epithelial cells and CD4(+) T-cells by HIV-1. 1204 57

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4+ T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics.
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PMID:Infectivity and replication capacity of drug-resistant human immunodeficiency virus type 1 variants isolated during primary infection. 1282 13

The initial virus strains from as many as 12% of individuals with primary human immunodeficiency virus (HIV) infection have a 50% inhibitory concentration </=0.4-fold that of HIV type 1(NL4-3) (HIV-1(NL4-3)) to ritonavir (hypersusceptibility [HS]). There is also substantial variation in replicative capacity (RC) or an in vitro assay of the contributions of protease (PR) and reverse transcriptase to viral fitness. In chronically infected antiretrovirally treated patients, amprenavir HS has been associated with the mutation N88S in PR, but this mutation is not seen in untreated patients. In this study, virus strains from 182 cases of primary HIV infection were analyzed, and a highly significant association between HS and low RC (</=10% that of HIV-1(NL4-3)) was observed (P < 10(-6)). Multivariate analysis was used to determine the genotypic basis of ritonavir HS, analyzing all polymorphic amino acid sites and insertions from p7gag through PR. Decision tree models developed on the entire Gag-plus-PR data set and on PR alone gave overall correct classifications of 73 and 72%, respectively, on cross-validation. They were also able to predict low RC, with sensitivities of 69 and 62% and specificities of 84 and 70%, respectively. The analysis shows that ritonavir HS in untreated primary HIV infection is not associated with single mutations but with combinations of amino acids at polymorphic sites and that the same genotypes which confer HS to PR inhibitors confer low RC. This supports the view that variation in PR function is directly responsible for variation in fitness among strains in primary infection.
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PMID:Genetic basis of hypersusceptibility to protease inhibitors and low replicative capacity of human immunodeficiency virus type 1 strains in primary infection. 1496 20

Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against human immunodeficiency virus type 1 (HIV-1). However, the antiviral mechanism of prostratin is not well defined. In the present study, we report that prostratin exhibits potent antiviral activity against different strains of HIV-1 (subtypes B and D), a clinical HIV isolate (L1), HIV-2 (ROD and EHO) and SIV (MAC251) with EC50-values ranging from 0.02-0.09 microg/ml. Prostratin was equally active against HIV strains resistant to the polyanionic binding inhibitor dextran sulphate, the fusion inhibitor T-20 (enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs). In contrast, prostratin lost 4.4- and 6.8-fold of its effect against the HIV strains resistant to AMD3100 and the quaternary ammonium salt QAS10+, respectively. As shown by time-of-addition experiments, prostratin needs to be present at the time of viral adsorption to exert its antiviral activity. We selected an HIV strain (NL4.3/PROS) resistant to prostratin in MT-4 cells. The sensitivity of NL4.3/PROS towards prostratin, dextran sulphate and QAS10+ was reduced by 3.2, 4.1 and >50-fold, respectively. However, NL4.3/ PROS was still sensitive to AMD3100, T-20, NRTIs (zidovudine and nevirapine) and a PI (ritonavir). Recombination of the gp160-gene of the NL4.3/PROS strain in a NL4.3 wild-type molecular clone fully rescued its phenotypic resistance. DNA sequencing of the NL4.3/PROS strain revealed mutations throughout the gp120 gene previously associated with resistance towards other HIV entry inhibitors. We concluded that prostratin inhibits the entry step of the replication cycle of HIV by interacting with a cellular target necessary for viral entry.
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PMID:Potent and selective inhibition of HIV and SIV by prostratin interacting with viral entry. 1496 38

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4(+)-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.
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PMID:Requirement for integrase during reverse transcription of human immunodeficiency virus type 1 and the effect of cysteine mutations of integrase on its interactions with reverse transcriptase. 1511 86

HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.
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PMID:Structural variability of the initiation complex of HIV-1 reverse transcription. 1519 85

In this study, we report on the efficacy of using a spleen necrosis virus (SNV)-based vector delivery system to block human immunodeficiency virus type I (HIV-1) replication in human hematopoietic cells. These efforts were directed towards the development of human immune system cell resistance to HIV-1 infection, based on the strategy of "intracellular immunization" via generation of a series of anti-HIV-1 therapeutic constructs carrying scFvs, single-chain variable fragments, against HIV-1 integrase and reverse transcriptase in combination with the trans-dominant mutant of HIV-1 Rev, RevM10. The efficiency of the anti-HIV-1 constructs were tested in viral challenge assays with different doses of HIV-1 NL4-3, Bal, 89.6 and R7-GFP strains. These experiments demonstrated the reduction of HIV-1 replication by these retroviral vector constructs in a range of 4- to 10-fold in CD4+ T-lymphocytes, human peripheral blood mononuclear cells (PBMCs), and primary human macrophages. We observed selective efficiency of SNV-based therapeutics in H9, C8166 and Jurkat T-lymphocytic cell lines, demonstrating the most efficient inhibition of HIV-1 replication in Jurkat T-cells. Thus, these data are the first demonstration of the ability of SNV-based retroviral vectors with select transgenes, which may have certain molecular advantages over other retroviral vector systems, to combat HIV-1 replication in human hematopoietic cells and support the potential for using SNV-expressed constructs in anti-HIV-1 molecular therapeutics.
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PMID:Spleen necrosis virus-based vector delivery of anti-HIV-1 genes potently protects human hematopoietic cells from HIV-1 infection. 1566 Nov 58


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